JP7175768B2 - Cell culture cassette and automatic device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cell culture cassette and an automated cell culture apparatus using the cassette. The invention thus relates to a device for cell culture called a cassette.

Among the fields using cell culture, cell therapy is the least industrialized. Therefore, there is a significant need to find techniques that can produce sufficient quantities of cells under optimal and safe conditions for use of these cells in therapeutic applications.

Some cell therapy processes require culturing or expansion of stem cells before they are re-infused into the patient because the amount collected is sometimes insufficient to have a therapeutic effect. It is essential to ensure the integrity of the therapeutic properties of the cells during culture. With current technology, the proposed solutions for ex vivo stem cell culture are skill-requiring, highly empirical, and ineffective.

In addition, current technology is unable to produce stem cells in sufficient quantities for therapeutic use. Therefore, there is a real need to develop bioreactor technology that has a compact geometry and is capable of growing cells in large numbers.

Automation of the stem cell expansion process for production applications in therapeutic or clinical settings requires cell production equipment that can operate in a controlled active area (clean room) with minimal human intervention. The stem cell culture process involves multiple steps to obtain these cells from a patient sample: isolating the cells, purifying them prior to culturing and final collection, and culturing them prior to sending them to a clinical treatment center. It is relatively complex as it involves the subsequent steps of purifying and packaging the cells.

All these processes must be performed according to manufacturing standards that impose strict sterility and traceability requirements. As the manufacturing process, which is often manual, increases, it becomes necessary to use different classes of containment areas to maintain the sterility of the process. Proper implementation of preparatory processes before and after culture often involves movement of process intermediates from one area to another, increasing both the risk of loss of sterility and the risk of mixing production flows.

For example, cell culture systems such as those described in US Patent Application Publication No. 2012/0308531 are known. Production of continuous cultures for stem cell production is performed by a complex system of subassemblies that transfer different reagents, cells, and gases to the reactor.

The applicant has also proposed an automatic device for automatic cell culture in document WO2013/135817. When cell culture is performed in a single bag , its handling can be difficult due to its flexible structure.

It is an object of the present invention, inter alia, to provide a simple, effective and economical solution to the above-mentioned prior art problems.

To this end, the present invention proposes a cell culture cassette, said cell culture cassette comprising:
- an at least partially rigid housing defining an interior space in which a cell culture bag defining an interior volume is placed and secured;
- conduits each connected at one end to the interior volume of the bag and each having a second end located outside the housing; and - a valve attached to the housing for opening and closing fluid flow through the conduits. Prepare.

Bags and valves are operated through the cassette housing. Only the second end of the conduit needs to be directly manipulated. According to the present invention, the cell culture cassette or device incorporates all elements that may pose challenges in terms of sterility and that must restrict its direct manipulation. These elements are pre-assembled into a disposable cell culture cassette. After use, all used conduits, valves, and culture bags can be disposed of according to good practice rules. Preferably, by incorporating the flexible bag into an at least partially rigid housing, the bag is retained within the housing and is very easy to handle. Similarly, incorporating the valve into the housing may facilitate identification of the position of the valve, and may facilitate manipulation by the incubator control means, as will become apparent in the discussion below.

Solutions that incorporate valves into the fluid cassette as a means of shutting off the cassette lines often use only manually operable plastic "clamps" that weaken during the freezing process. By also avoiding the use of pinch solenoid valves that need to be installed with a tether, it makes it possible to improve the automation of the use of said cassettes in cell culture processes. This solution therefore avoids errors associated with manipulation of the conduit and incorrect insertion of the conduit into the solenoid valve.

Cassettes in which bags contain cell culture media can be frozen if desired.

Another feature of the invention is that the conduits are formed by flexible tubing and at least some of the valves are provided with movable clamping members for the tubing on structural elements of the housing. As described above, the incorporation of the valve into the cassette allows independent and reliable maintenance of the closed circuit (storage or incubation time) for the desired period of time without the need for external energy.

According to yet another feature of the invention, each moveable member has an axis of rotation, and the radial periphery of each moveable member comprises at least one helical surface about the axis of rotation, the helical surface comprising: A clamping surface of the tube is formed such that rotation of the member alters the passage cross-section of fluid through the tube.

Advantageously, the valve can maintain a given position. More specifically, the valve is designed so that the movable member can maintain a given open or closed position without the supply of energy. In particular, this can be achieved by having the perpendicular to the point of contact between the helical surface and the tube oriented towards the axis of rotation of the movable member so that the torque about said axis is zero.

In one particular design, the movable members are mounted in recesses in a plate fixed to the housing, each recess receiving a movable member. If the housing is formed of two parts, a lower half-shell and an upper half-shell, attaching the cell culture bag to the lower half-shell and attaching a movable member for clamping the tube to the lower half-shell. , and can be fitted with a plate that allows it to hold the movable member at the same time. The plate can for example be attached to the lower half-shell, for example by screws. The plate therefore makes it possible to reliably and easily assemble the various parts mentioned above. This ensures that the valve is maintained.

Each movable member comprises an end accessible from outside the housing, said end being adapted to co-operate with second rotary coupling means of the actuator, which may be electromechanical. Have the means.

Access to the movable member of the valve from the outside allows a second coupling means, e.g. It is possible to attach the cassette to a suitable support of the incubator provided with the coupling means. This external accessibility allows manual operation of the valve in the event of a failure of the valve drive means.

Preferably, the movable member is rotationally centrally guided so that one end thereof is in the housing opening and the opposite end is in the plate opening. If the housing consists of two half-shells, the plate can be fixed inside the housing between these two half-shells.

According to another feature of the invention, the pouch comprises a first flexible wall and a second flexible wall which are substantially parallel to each other in both empty and liquid-filled states. It has a rectangular shape.

Since the rectangular shape is substantially flat, it is measured between the first wall and the second wall in a first direction substantially perpendicular to the first wall and the second wall. The thickness is very small, ie much smaller than the other two dimensions of the bag measured in the other two directions in space relative to each other and perpendicular to the first direction. "Very significantly lower" means at least 1/10 as commonly accepted in mathematics.

Preferably, the ratio of the sum of the surfaces of the first wall and the second wall to the total internal volume of the bag is between 500 and 690 cm ² /L, preferably of the order of 580 cm ² /L. This ratio ensures optimal heat exchange between the flexible bag and the outside when the cassette is placed inside an incubator within a constant temperature enclosure.

The distance between said first wall and said second wall measured in a direction perpendicular to the first and second walls or the thickness of the pouch in the liquid-filled state is advantageously 20 mm less than, preferably 10 to 20 mm, more preferably about 14 mm. The use of a small thickness further promotes heat diffusion in the liquid of the bag. It also allows uniform freezing and thawing of the various components of the culture medium, which is more rapid at constant temperature.

The bag according to the invention makes it possible to maintain the stability of the culture medium during the cryogenic freezing process. Indeed, the high freezing rate of blood-derived biological products is a guarantee of preservation of components, homogeneity of the medium after thawing, and the absence of concentration effects of salts and proteins in solution in the center of the frozen mass. Become. The bag according to the invention facilitates rapid freezing of the culture medium due to its specific shape with high surface area/volume ratio and low thickness.

According to another characteristic of the invention, the walls of the bag are permeable to gases, in particular _CO2 , and preferably have the property of limiting adhesion of cells to the walls of the bag as much as possible.

The pouch is preferably flexible, ie deformable. The pouch preferably comprises flexible, gas permeable walls capable of retaining the liquid culture medium and its cell expansion. In fact, it is a question of retaining any liquid contained within the bag. The bag preferably has excellent oxygen and carbon dioxide permeability, which allows good ventilation of the contents of the bag without opening the bag and thus without the risk of contamination of the contents. Become. In one particular embodiment of the invention, the bag has the following permeation properties (permeation volume per day (cm ³ ) at 37° C. and under a pressure of 1 atmosphere, i.e. 1 bar (100 kPa)): 418-508, 699-1200 for carbon dioxide, 157-200 for nitrogen, and 0.05-0.1 for water.

The bag is formed, for example, from a thin film that can be made from a fluoropolymer, especially a copolymer such as fluorethylene propylene, having a thickness of about 120 pm. In one preferred embodiment, the bag has a substantially rectangular shape with a small thickness and can reach 230-250 mm in width in the flattest state and 230-250 mm in length in the flat state. .

The use of materials belonging to the fluoropolymer family also has the advantage of being gas permeable and having low adhesion to liquids.

The housing preferably has a shape substantially corresponding to the shape of a cuboid and can comprise a lower half-shell and an upper half-shell fixed to each other, the lower half-shell being rigid and the The upper half-shell is rigid or flexible.

The housing can thus also have a substantially flat shape with a very small thickness compared to its length and width.

The housing can be formed from any elastomeric thermoplastic material such as SEBS (styrene-ethylene-butadiene-styrene). The lower half-shell may be rigid to ensure optimal retention of the flexible bag and the upper half-shell may be partially rigid and provide grip and some degree of deformability. It may comprise one or more flexible regions formed from an elastomeric material to facilitate and facilitate attachment of the cassette to the support and agitation means described below. Of course, the upper half-shell can also be completely rigid like the lower half-shell.

Both the lower and upper half-shells can be assembled by clipping, gluing or welding their perimeter edges. The half-shells may have the same thickness or may have different thicknesses.

In one preferred embodiment of the invention, the upper half-shell can be provided with a central opening or recess, the shape and size of which is such that the liquid-containing cassette is oscillated about a horizontal axis. designed to allow waves of liquid in the bag to propagate to the opening when the bag is closed, the opening being configured upwardly. Wave formation is desirable to achieve rapid homogenization of the liquid within the bag. Therefore, the formation of recesses, sometimes called cutouts or openings, does not increase the thickness of the housing. This recess also ensures better diffusion of the gas in contact with the bag. The recess may have dimensions of about 210 mm wide and about 28 mm long.

Preferably, the lower half-shell is provided with a plurality of holes, preferably with a diameter of eg less than 20 mm. The integration of the holes facilitates the movement of gas in the same manner as the openings in the upper half-shell while the bag remains securely held in the lower half-shell.

In one practical embodiment of the invention, the lower half-shell may comprise approximately 50-400 holes each having a diameter of the order of 5-20 mm, eg 10 mm. The holes can be distributed according to the mesh size in a basic pattern that is a pattern of equilateral triangles with a unit length of 5-40 mm, eg 20 mm.

In the cassette design according to the invention, the conduit extends through one of the outer perimeter edges of one of the lower or upper half-shells. The conduit can be received in a slot in one of the outer perimeter edges of the lower or upper half-shell.

The cassette preferably comprises a conduit support strip located outside the housing through which at least some of the conduits pass. This strip supports a conduit extending outside the cassette housing and limits unintentional movement of the free end of the conduit. In addition, the support is intended to form a waterproof passageway element of the incubator door, as will become more apparent in the following description.

The cassette preferably contains cell culture medium stored in a bag, which can be frozen.

A cell culture medium can be a cell culture medium whose composition is suitable for the growth of mammalian cells, particularly human cells, excluding human embryonic stem cells. The culture medium may or may not contain cells in culture.

These cells, in particular human cells other than human embryonic stem cells, may advantageously be or comprise (human) CD34+ cells, in particular (human) CD34+ hematopoietic cells or (human) non-hematopoietic CD34+ cells. It's okay. These (human) CD34+ cells can be, for example, cells from (human) peripheral blood, or (human) umbilical cord, or human tissue, in particular (human) peripheral blood.

These cells, in particular human cells, with the exception of human embryonic stem cells, may advantageously be or comprise stem cells or progenitor cells, in particular multipotent or pluripotent cells.

These cells are not and do not contain human embryonic cells.

The cell culture medium may contain components that allow the proliferation of these cells, in particular components that allow their proliferation while maintaining them in an undifferentiated state, or at least without reaching a stage of terminal differentiation. .

This cell culture medium may contain insulin, transferrin and (human) plasma or serum.

The cell culture medium may contain or lack feeder cells such as fibroblasts.

The cell culture medium may be or include, for example, Iscove's Modified Dulbecco's Medium (IMDM), optionally supplemented with glutamine or glutamine-containing peptides: see, for example, Iscove, N.N. and Melchers, F. J. Exper. Med. 147:923 (1978).

In particular, the cell culture medium may be or comprise IMDM medium, optionally supplemented with glutamine or glutamine-containing peptides, which medium is free of fibroblasts and human (recombinant ) insulin, (human) transferrin and (human) plasma or serum.

For example, the culture medium is:
1-50 μg/mL, especially 8-12 μg/mL insulin,
• 100-2000 μg/mL, especially 300-500 μg/mL transferrin, and • 1-30%, especially 4-12% (human) serum or plasma. The culture medium may also contain heparin, eg, 1.5-3.5 IU/mL heparin.

The culture medium may also contain erythropoietin, including human (recombinant) erythropoietin.

The culture medium contains hydrocortisone, SCF (stem cell factor), interleukin 3 (IL-3), thrombopoietin (TOP), FLT3 (Fms-like tyrosine kinase 3), BMP4 (bone morphogenetic protein 4), VEGF-A165 (vascular endothelial proliferation One or more growth factors may also be included, including factor A165), and one or more growth factors selected from IL-6.

The culture medium is, but not limited to,
- hydrocortisone, or - SCF, or - SCF and IL-3, or - hydrocortisone and SCF, or - hydrocortisone, SCF, and IL-3, or - SCF, TOP, FLT3, BMP4, VEGF-A165, IL-3, and IL-6.

The culture medium originates in particular from WO 2011/101468 (or this PCT International Application incorporated herein by reference, such as in particular US Patent Application Publication No. 2013/0078721 A1, or one of the US patent applications based thereon).

The cassette may contain culture medium and/or cells as described above.

According to one method of production, the cassette, especially in frozen form, contains culture medium in a bag, but no cells.

According to another production method, the cassette contains culture medium in a bag, especially in non-frozen form, and also cells, especially cultured cells, especially (human) CD34+ cells as described above.

The present invention also relates to a cell culture incubator, comprising a thermostatic enclosure and a device for supporting and agitating the cell culture cassette defined above, and the cell culture cassette provided within the support device. means for locating and blocking the position of

In one embodiment, the support and agitation device comprises a support tray for the cell culture cassette, pivotally mounted about a horizontal axis, for agitating and homogenizing the contents of the bag. oscillate about the horizontal axis.

The support and agitation device may comprise a connecting rod, one upper end of which is connected to the tray via an oscillator, and a lower end of which is a motor having a substantially horizontal axis. is connected to a crank handle that is rotatably driven by the shaft of the

The incubator may comprise means for controlling the position of the cell culture cassette in the support means so that it is properly positioned on the support means and ready for use.

The tray may hold an electromechanical actuator for opening and closing the valve, one output of which cooperates with the first coupling means of the valve of the cassette when the cassette is in said given position. It holds a second coupling means adapted to work.

According to another feature of the invention, the incubator includes a door for sealing an opening in the enclosure, the opening having a peripheral edge adapted to receive the conduit support strip. A shaped recess is formed and the strip forms a sealing member between the door and the perimeter edge when the door is in the closed position of the enclosure.

The incorporation of conduit support strips allows for quick and easy placement of conduits at the inlet. Indeed, in contrast to known techniques which consist of incorporating grooves in the housing of the conduits directly into the outer perimeter and placing the conduits one by one in the grooves, the present invention attaches all the conduits in a single step. Suggest. In addition, the sealing is no longer done by the conduit, but by the strip, which makes it easier to make the seal because the strip can have a rigid structure that can support the door.

The present invention also provides at least one incubator of the type described above, and for regulating culture conditions in an enclosure of said at least one incubator and for controlling valves of a cassette housed in said enclosure. It also relates to an automated cell culture apparatus, comprising a computer control system including means for acquiring and recording .

The present invention also relates to a process for culturing cells with an automated device as described above, which process comprises:
(a) placing the cell culture cassette described above on a support and agitation device in the thaw position within the enclosure of the incubator;
(b) supplying the bag with cells to be cultured;
(c) placing the cassette on the support and agitation device in a cell culture position;
(d) shaking the cell culture cassette for a period of time to homogenize its contents;
(e) maintaining the cell culture cassette under incubation conditions for several days; and (f) collecting the contents of the cassette by one of the tubes of the cassette.

This process:
- During step (d), further comprising one or more steps of sampling the contents of the bag, wherein prior to each of said one or more steps, the support tray is placed in a horizontal culture position to sample said bag; A step of tilting to a tilted position where the collection area is the lowest point of the bag is performed.

The invention will be better understood and other details, advantages and features of the invention will emerge from the following description, given by way of example only, with reference to the accompanying drawings in which: FIG.

1 is a schematic perspective view of an automated cell culture apparatus according to the present invention, comprising an incubator defining an open thermostatically controlled enclosure; FIG. FIG. 2 is a schematic perspective view of the incubator in isolation. Figures 3A and 3B are schematic side views of the support and agitation means and the cassette. Figure 4 is a schematic perspective view of the support and stirring means and the cassette. FIG. 5 is a schematic top view of a cassette supported by support and stirring means; Figures 6A-6C show different operating positions of the cassette in the thermostatic enclosure of the automated device. Figure 7 is a schematic perspective view of a cassette according to the invention showing the upper half-shell; Figure 8 is a schematic perspective view of a cassette according to the invention showing the lower half-shell; FIG. 9 is a schematic enlarged view of the dotted line area in FIG. Figure 10 is a schematic perspective view of the cassette of Figure 7 with the upper half-shell removed; FIG. 11 is a schematic perspective view of the separated lower half-shell. 12 is a schematic enlarged perspective view of the area enclosed by the dotted line in FIG. 11. FIG. FIG. 13 is a schematic perspective view of the upper half-shell; FIG. 14 is a schematic perspective view of the upper half-shell. FIG. 15A is a schematic perspective view of a receiving plate for opening and closing the valves of the conduits of the cassette; Figures 15B and 15C are schematic enlarged perspective views of only a portion of the plate shown in Figure 15A. Figures 16A, 16B and 16C show a first variation of a valve that can be used with the plate of Figure 15A. Figures 17A and 17B show a second variation of the valve that can be used with the plate of Figure 15A. Figure 18 is a schematic perspective view of means for rotating the valve of Figures 16A, 16B and 16C. Figures 19A and 19B are schematic perspective views of the valve actuator of Figures 17A and 17B. FIG. 20 is a schematic perspective view of a cell culture system consisting of multiple incubators and centrifuges. FIG. 21 is an organization chart showing the steps of the cell culture process according to the invention.

Reference is first made to FIGS. 1 and 2, which represent an example of one embodiment of an automated cell culture apparatus 10 according to the present invention. This automated device 10 is intended, for example, for culturing stem cells, with the exception of human embryonic stem cells.

As shown, the automatic device 10 consists essentially of three elements:
- an incubator 12 with a thermostatic enclosure comprising a device 14 for supporting and agitating the cell culture cassettes 16 according to the invention (Figs. 1 and 3);
a rack 18 that supports the incubator 12 and centrifuge 20;

Typically, the computer system includes data entry and recording equipment, data processing equipment, display equipment, and signal transmission control and signal transmission monitoring equipment for the incubator and cassette 16 valves and pumps. Preferably, the computer system includes a touch screen display 22 and operator or user data entry.

Control and traceability of biological protocol steps can be ensured by computer systems and suitable human-machine interfaces (HMIs), which allow, inter alia:
Full traceability of the manufacturing process for each stem cell transplant, i.e. automated equipment, operator intervention to culture parameters such as temperature, _CO2 rate and time for each step, as well as verification required at specific process steps. guarantee work,
Ensuring traceability of consumables and reagents used in the process and verification of the integrity of information collected in internal databases by means of input interfaces, means for reading and storing barcode data, and RFID labels; . This traceability can be applied to the entire manufacturing, storage, and transportation chain of consumables and reagents, as well as the condition of materials for maintaining the temperature of these components.
- systematically showing the user the details of the steps of the protocol through videos and images illustrating the progression of the steps of the process;
- securely recording patient identification data, biological data associated with the characterization of stem cell grafts;
- to control each actuator control function;
• Recording and interpreting data from various sensors present in automated devices or fluid cassettes.

As shown in FIG. 2, the incubator 12 has two doors, a first inner door 22 abutting a first contact surface 24 surrounding an opening 26 in an enclosure 28 of the incubator 12, and the It comprises a second outer door 30 abutting a second contact surface 31 surrounding the first contact surface 24 . The first door 22 and the second door 30 are each, for example, pivotable about the same vertical axis. Enclosure 28 houses ventilation means 33 located on top of enclosure 28 (FIGS. 1, 6A, 6B and 6C). These ventilation means 33 are designed and constructed to allow uniform distribution of temperature and gases within the incubator chamber necessary for efficient cell culture and to have the appropriate temperature (Fig. 1, 6A, 6B, and 6C). Thus, during thawing of the culture medium contained within the bag, the heat within the chamber is evenly distributed to enable rapid thawing of the cell culture medium.

Inner door 22 may be formed of clear glass to allow viewing of the contents of chamber 28 of incubator 12 while keeping incubator 12 in a closed position. The opening 26 of the enclosure 28 has an outer peripheral edge 24 corresponding to the first contact surface, the lower edge 24a of which is the tube or the It comprises a recess 32 adapted to receive a strip 34 for holding conduits 36,38. The bar 34 visible in Figures 4, 5, 7 and 8 forms a rigid shell which may have a substantially parallelepiped shape. The bar 34 may also have two opposing sides 40 connected together by a lower surface 42 and an upper surface 42 . Conduits or tubes 36 , 38 extend through holes in strip 34 in a watertight manner. These holes are formed in walls 44 that are substantially perpendicular to the sides 40 and the lower and upper walls 42 . Each side 40 of the bar 34 has a groove 46 parallel to the bottom 42 and top 42 surfaces. The grooves 46 in the sides 40 are arranged asymmetrically with respect to the center plane of the bar 34 and parallel to both sides 40 . These grooves 46 are adapted to receive ribs (not visible) extending from the sides of the recesses 32 of the lower peripheral edge 24a of the incubator 12 so that the strips 34 are positioned within the recesses 32. there is The asymmetrical arrangement of grooves 46 allows for the implementation of coding when strips 34 are attached to recesses 32 of incubator 12 . More generally, the strip can therefore be provided with coding means when attaching it to a recess in the outer periphery of the opening of the incubator. The opening may then be closed by a movable member such as a door or hatch that is movable between an open position of the incubator and a closed position of the incubator. Bar or rigid shell 34 preferably has a shape complementary to the shape of recess 32 . This complementary shape is such that when the strip 34 is in place within the recess 32 , its lower surface 42 contacts the bottom surface of the recess 32 and its upper surface 42 contacts the outer periphery of the opening 26 of the enclosure 28 of the incubator 12 . It may be coplanar with the remainder of 24 to form a substantially flat surface capable of sealingly supporting the first door 22 . In this way, the sealing of the inner door 22 against the outer edge 24 of the opening 26 of the enclosure 28 of the incubator 12 can be achieved in a simple manner. In addition, passage of tubes 36, 38 or conduits through first door 22 can be easily accomplished without having to manipulate the tubes 36, 38 one by one. Finally, when the first door 22 abuts the strip 34, the closing force is applied to the rigid structure of the strip 34 rather than to the tubes 36, 38, thereby preventing the tubes 36, 38 from collapsing. , these fluid passage cross sections are ensured. Preferably, the recess 32 is provided with a presence sensor 47 for the strip 34 to confirm the correct placement of the strip in the recess 32 so that the first door 22 and then the second door 30 can be closed. can be done.

As shown in FIG. 2, the front face of incubator chamber 12 has another recess 48 that connects to recess 32 in outer rim 24 of opening 26 of enclosure 28 . This recess 48 is adapted to allow conduits or tubes 36, 38 to pass through recesses 32 in first door 22 and second door 30 to the centrifuge. In practice, this second recess 48 can be closed by a hatch (not shown), for example a sliding hatch.

As shown in FIG. 3A, the isothermal enclosure 28 includes a device 14 for supporting and agitating the cell culture cassette 16 according to the invention. The device can be divided into a supporting device 50 and a stirring device 52 which are connected to each other. The support device 50 consists of a support tray 54 or cassette 16 support cradle and a base 60 . The tray 54 has a first end 56 located at the bottom of the isothermal enclosure 28 and a second end 58 opposite the first end, the second end 58 comprising: Pivotably about a horizontal axis 63 is connected to a substantially vertically extending base 60 which is retained by a wall 62 forming the floor of the isothermal enclosure 28 (FIGS. 3A and 3B). Four). More precisely, as shown in FIG. 3B, the first end 56 of the tray 54 has two arms 65 formed on either side of the tray 54 extending vertically from the floor 62 of the enclosure 28. It is held by an upper shaft 63 and rotated around the shaft 63 .

The agitator 52 comprises a connecting rod/crank system with connecting rods 64, one upper end of which is an oscillator or vertical which supports the tray 54 on its lower surface at its opposite upper end. It is connected to the lower end of the vibrating rod 66 . The lower end of connecting rod 64 is connected to crank handle 68 which is driven in rotation by a motor shaft having a substantially horizontal axis of rotation 70 . This axis 70 is perpendicular to axis 63 . The upper end 66 of the oscillator is connected to the tray so as to allow movement of the oscillator in a plane perpendicular to the axis of rotation 70 . The upper end of the oscillator 66 is also movably connected to the support tray 54 in the direction of the axis 70 , and this connection optionally moves the fingers from the upper end of the oscillator 66 into slots 71 or grooves of the tray 54 . It can be done by sliding it in.

As shown in FIGS. 3, 4 and 5, oscillator 64 passes through floor wall 62 of enclosure 28 of incubator 12 . Sealing means, such as bellows, are preferably provided at the level of the passage area of the floor wall 62 to avoid the transfer of heat and moisture outside the isothermal enclosure 28, which could damage the mechanical components of the agitation device 52. is provided around the oscillator 66 at .

Figures 6A, 6B and 6C show the three positions that the tray 54 assumes during its movement by the stirring means. FIG. 6A shows the tray 54 in its upper position and FIG. 6B shows the tray 54 in its lower position. In either of these two positions the tray is inclined with respect to the horizontal plane. FIG. 6C shows a central position in which the tray 54 is substantially horizontal. Thus, during operation, cassette support tray 54 can be tilted between its extreme positions (FIGS. 6A and 6C) about axis 63 (FIG. 4).

As shown in FIG. 2, support tray 54 of cassette 16 is formed by a frame 72 having an opening 74 or hollow portion and a solid portion 76 . A hollow portion 74 adjacent the first end 56 of the tray 54 faces the lower half-shell of the cassette described below. Solid portion 76 is positioned near second end 58 of tray 54 . This solid portion 76 serves to position and lock the cell culture cassette in a given position on the tray 54 corresponding to the position at which the cassette 16 is supported on the frame 72 and allows the incubation step to take place. have the means. The positioning means comprise two pins 78 projecting upwardly to cooperate with two blind holes in the cassette 16 (described below). Means for locking the cassette 16 include clip hooks 80 or snap hooks that engage cassette hooks, described below, to lock the cassette 16 to the tray 54 . A hook 80 on the solid portion 46 of the frame 72 of the tray 54 is positioned between two pins 78 .

As shown in FIG. 2, the tray includes three electromechanical actuators 82 for opening and closing the valves, each actuator 82 corresponding to the position of the cassette 16 when it is supported on the frame 72 of the tray 54. An output is provided having second coupling means cooperating with the first valve coupling means of the cassette.

A structure 84 is positioned above the frame 72 to support the cassette 16 away from the frame 72 . This support structure 84 makes it possible to support the cassette 16 in a second position corresponding to the thawing phase. This support structure 84 comprises two transverse fins 86 connected to each other by flat walls 88, together forming a common support plane for the cassette 16 in the second position. Note that the support surfaces of the cassette 16 in the first position and the support surfaces of the cassette 16 in the second position are inclined with respect to each other. The second position is intended to support the cassette 16 vacuum sealed within the envelope, away from the frame 72 to avoid any tearing of the envelope, which is generally made of plastic, by pins 78 during the thawing step. to enable.

Referring now to FIGS. 7 and 8, these figures substantially correspond to the shape of a cuboid having a first end 92 and a second end 94 referenced in positioning the cassette 16 relative to the tray 54. Fig. 2 shows a cell culture cassette 16 according to the invention with a shaped housing 90; The housing 90 has a lower half-shell 96 (FIGS. 9-12) and an upper half-shell 98 (FIGS. 13-14) integral with each other, the lower half-shell 96 being rigid and the upper half-shell 98 is rigid or partially rigid, ie includes a flexible portion. Together, the lower half-shell 96 and the upper half-shell 98 define a housing in which is secured a flexible cell culture bag 100 defining an interior volume to be filled with cell culture medium.

The lower half-shell 96 and upper half-shell 98 are connected to each other by four peripheral edges 102a, 102b. The two lower half-shells 96 and upper half-shells 98 can be assembled by clipping, gluing or welding their perimeter edges 102a, 102b. In one preferred embodiment, both the lower half-shell 96 and the upper half-shell 98 are rigid.

The lower half-shell 96 is provided with a plurality of holes 104 preferably having a diameter of less than 20 mm. For example, cassette 16 is 405 mm long, 305 mm wide and 25 mm thick. Cassette 16 also includes two spaced apart blind holes 106 with through openings 107 formed therebetween. A blind hole 106 is formed by a recess 109 in the lower half-shell 96 extending towards the upper half-shell 98 (FIGS. 11 and 12). The upper half-shell 98 has an opening shaped and sized to allow propagation of a wave of liquid to the opening of the bag 100 when the liquid-containing cassette 16 is vibrated about the horizontal axis 63. A portion or recess 108 is provided, the opening 108 (or cutout) being oriented upwards (FIGS. 7, 13, 14). As described above, the formation of recess 108 is such that when cassette 16 is mounted in a second position on tray 54, the tray moves in an oscillating fashion as depicted in FIGS. 6A, 6B, and 6C. Allows the surface of the bag 100 within the housing to ripple when pressed. The upper half-shell 98 is provided with elastic tongues 110 holding clip hooks 112 which engage hooks 80 of the frame 72 of the tray 54, as already described above. Accordingly, hooks 112 of upper half-shell 98 are configured to pass through openings 107 of lower half-shell 96 . Finally, the upper half-shell 98 also includes a circular hole 114 for inserting a manual valve actuation plug 116, the use of which will become more clearly apparent with reference to FIGS. deaf.

Peripheral edge 102a of lower half-shell 96, located at first end 92 of housing 90, extends when cell culture cassette 16 is mounted in its first position on tray 54 within enclosure 28 of incubator 12. , has linear ribs 116 adapted to cooperate with slots 118 in the support structure 84 of the tray 54 (FIGS. 8, 9, 11, 12). In this first position, the clip hooks 112 of the upper half-shell 98 pass through the openings 107 of the lower half-shell 96 and cooperate with the hooks 80 of the frame 72 of the tray 54 so that the cassette 16 is held in this first position. position (Figures 3, 4, and 5). To release the cassette 16 and remove it from the culture enclosure 28 , simply press the tab 110 , which disengages the hook 110 of the upper half-shell 98 from the hook 80 and thus the tray 54 .

A pad 120 is secured by snapping into the upper half-shell and is suitable for holding the tubes 36, 38 of the cassette 16 in a folded position on the outer surface of the upper half-shell 98.

FIG. 10 shows the interior of housing 90, with upper half-shell 98 not shown. As can be seen, the cell culture bag 100 has a substantially rectangular shape with a first wall 122 and a second wall (not visible) that are substantially parallel to each other in an empty state and a liquid filled state. have a shape. The first wall 122 and the second wall are connected together by their perimeter edges 123 using any means such as welding or gluing. In one particular embodiment of the invention, the ratio of the surface area of one of the first wall 122 or the second wall to the internal volume of the bag is between 540 and 600 cm ² /L, preferably 580 cm ^{2 .} /L. The distance between the first wall 122 and the second wall is less than 20 mm, preferably 10-20 mm, more preferably about 14 mm.

Cell culture bag 100 is fixed to lower half shell 96 by pins 124 projecting from the inner surface of lower half shell 96 . These pins 124 pass through holes in the peripheral edge 123 of the cell culture bag 100 . The interior volume of bag 100 communicates with the outside via multiple conduits. In particular, the cassette 16 can comprise four conduits or flexible tubes 36, 38, three of which are attached to the already described bar 34 and the fourth conduit 38 is independently there is

More specifically, cassette 16 includes first and second sampling tubes 36a, 36b and an exhaust tube 36c. Tube 38 corresponds to a tube for injecting cells of interest. As shown in Figures 5, 7, 8, 9 and 10, the cassette also includes a tube 39 for initial filling of the bag 100 with culture medium. In one preferred configuration of cassette 16 according to the present invention, four tubes 36a, 36b, 36c, 38 are provided with connectors, which may be sterile connectors and/or needle-free withdrawal connectors, which connectors are , and if necessary, also provided with non-return means. In the following description, when reference number 36 is used alone, this reference number 36 refers to at least one of tubes 36a, 36b, or 36c.

As shown in FIG. 10, the tubes 36, 38 pass through the same outer peripheral edge 102a of the lower half shell 96, i. extended. Fill tube 39 extends on one side adjacent to the side through which tubes 36, 38 pass, and in the portion of tube 36 where tube 39 is arranged to provide fluid communication between valve 128 and bag 100, It is connected to the end of tube 38 located within housing 90 .

Each tube 36, 38 passes through a valve 128, 130a, 130b, 130c for passing/blocking fluid flow through the tube 36, 38, the pinch valves 128, 130 being located within the housing 90. It is In the example shown, three tubes 36 pass through a first type of valve 130, tube 36a through valve 130a, tube 36b through valve 130b, and tube 36c through valve 130c. Meanwhile, the fourth tube 38 passes through a second type valve 128 (Figs. 10 and 15A). More precisely, the housing 90 comprises a plate 132 (Figs. 15A, 15B and 15C) with movable members 136, 138 (Figs. 16 and 17) for clamping the tubes 36, 38 respectively. It has a plurality of substantially cylindrical recesses 134 for receiving. This plate thus forms the body or fixed part of the valves 128,130. Each recess 134 in plate 132 has an annular bottom wall 140 defining a central hole 142 that aligns with hole 144 in lower half-shell 96 when plate 132 is installed in housing 90 (FIGS. 10 and 10). 12).

Each movable member 136, 138 has a lower end 136a, 138a and an upper end 136b, 138b with respect to the lower half shell 96, the upper half shell 98 and the placement of the cassette in the vertical direction. Of course, it would also be possible, more generally, to have the lower end as the first end and the upper end as the second end opposite the first end. Accordingly, the upper end 136b, 138b of each movable member 136, 138 extends a cylindrical wall 148b adapted to fit into the hole 142 of said recess around the hole 142 of the recess 134 of the plate 132, with an axis of rotation 136c, It has an annular shoulder 146b that supports and surrounds along 138c. In this case, surface 146b of member 136 bears against bottom surface 140 of recess 134 of valves 130a, 130b, 130c. Accordingly, it is possible to guide the second ends 136b, 136a of the movable members 136, 138 in the recess 134 of the plate 132 by rotation. A lower end 136a, 138a of each movable member 136, 138 includes an annular shoulder 146a adapted to support around an aperture 144 in the lower half shell 96 along an axis of rotation 136c, 138c and the lower half and a cylindrical wall 148a adapted to be inserted into the bore 144 of the shell 96. In this case, surface 146b of component 138 rests against bottom surface 140 of recess 134 of valve 128. FIG. In this way, each movable member 136, 138 is rotationally centered relative to the lower half-shell 96 and the plate 142, which itself is a stud on the inner surface of the lower half-shell 96. The upper half-shell 98 secures its peripheral edge 102b to the peripheral edge 102a of the lower half-shell 96, through which 152 is inserted. Accordingly, the lower ends 136a, 138a of the movable members 136, 138 are exposed through the lower half-shell 96 and are therefore accessible from the outer surface of the lower half-shell 96 as shown in FIG. be.

16A, 16B, 16C in relation to the tube 36. At their lower end 136a, a second portion of the movable component 136 is positioned within the incubator. It has a first coupling means accessible from outside the housing for cooperation with the actuator coupling means 82 . The first coupling means formed on the underside of member 136 are here a radial slot 151 whose radial inner end traverses the axis of rotation 136c and a semi-circular groove centered on the axis of rotation 136c. Equipped with 153. The second coupling means of the actuator 82 may, for example, comprise two rods 155a, 155b, of which the first rod 155a is located at the center of the axis of rotation of the actuator 82 and the second rod 155b. are radially offset with respect to the first rod 155a. As shown in FIG. 3A, when the cassette 16 is mounted in the frame 72, the first rod 155a engages the radially inner end of the slot 151 and the second rod 155b extends into the semi-circular groove. Engage 153. It should be appreciated that the use of semi-circular groove 153 ensures that second rod 153b cooperates with groove 153 regardless of the angular position of movable member 136 prior to mounting cassette 16 to frame 72 . The integration of radial slots 151 into movable member 136 allows manual actuation of movable member 136, if desired.

In the case of the valve 138 of FIGS. 17A and 17B associated with the tube 38, the first coupling means are formed at the upper end 138b of the movable member 138. As shown in FIG. Unlike the three movable members 136 of valve 130, the movable members 138 of valve 128 are not actuated by electromechanical actuators, but are manually operated by rotation of members or knobs 154 shown in FIGS. 18A and 18B. is operated by Here, the coupling is effected by a cross recess 157 in the upper end 138b of the movable member 138, into which a complementary cross member 156 of the actuating button 154 engages. A button 154 is secured to the upper half-shell 98 by a plurality of elastically deformable hooks 158 that engage the inner circumference of the circular hole 114 in the upper half-shell 98 .

To clamp the tubes 36,38, each movable member 136,138 is provided with a helical protrusion 160 about its axis of rotation 136c,138c. The radially outer surface of each projection 160 forms a bearing surface for the tubes 36,38. Each protrusion 160 has a plane of symmetry in a plane perpendicular to the axis of rotation 136c, 138c. Each tube 36, 38 is partially received within a semi-circular cross-section rectilinear groove 162 of the lower half-shell 96 and another portion is received within a semi-circular cross-section rectilinear groove 164 of the plate 132, thereby , passes through the outer periphery of the recess 134 of the plate 132 in a direction perpendicular to the axes of rotation 136c, 138c of the movable member (FIGS. 11, 12, 15A, 15B, 15C). Thus, rotation of the movable members 136,138 of the valves 128,130 ensures that the projections 160 progressively open or close portions of the tubes 36,38 depending on the direction of rotation of the movable members 136,138.

Each annular bottom wall 140 of the recess in plate 132 carries two stop members 166a, 166b projecting inwardly of recess 134 and spaced circumferentially from each other. A notch 167 is formed in each annular bottom wall 140 of the recess 134 so as to define in the recess 134 an elastic lamella 168 having a free end which retains the projections, which are attached to the movable members 136,138. 15B, 15C). The projection 170 slidably cooperates with a groove 172 of each movable member 136, 138 having an arcuate concave curved bottom. 16A, 16B, and 16C, the groove 172 is a radial annular flange 174 axially disposed between the protrusion 160 and the upper annular shoulder 146b. It is formed on the surface of 136 facing the upper end portion 136b. Similarly, in the case of the movable member 138 of FIGS. 17A and 17B, the groove 172 is a radial annular flange 174 axially disposed between the protrusion 160 and the upper annular shoulder 146b. It is formed on the surface facing the upper end portion 138b.

In order to form the rest positions of the movable members 136, 138, i.e. one position corresponding to an open position through which fluid can flow and another position corresponding to a closed position in which fluid cannot flow, a semi-spherical cavity 176a is formed. , 176b are formed at the circumferential ends of each groove 170,172. Also note that each movable member 136, 138 includes a radial finger 178 extending laterally from the upper shoulder 146b, the finger 178 contacting the face of the flange 174 having the groove 172 therein. want to be Thus, in the position of blocking flow through tubes 36, 38, moveable members 136, 138 are positioned such that projections 170 of resilient lamellae 168 engage cavities or recesses 176a of moveable members 136, 138, and the The radial fingers 178 of the movable members 136, 138 are arranged to stop counterclockwise rotation in FIG. 15 against the stop member 166a. In the position of passage of flow through tubes 36, 38, movable members 136, 138 are positioned such that protrusions 170 of resilient lamellae 168 engage cavities or recesses 176b of the movable members 136, 138 to The radial fingers 178 of 136, 138 are arranged to stop on clockwise rotation in FIG. 15 relative to the stop member 166b. It should be appreciated that the cooperation of the protrusion 170 and the cavities 176a, 176b makes it possible to ensure resilient locking of the movable members 136, 138 in the closed and open positions.

It will be readily appreciated that the valves described above may well be used in other devices requiring a device for passing/blocking fluid flow through the manifold. Accordingly, the present description includes any device incorporating the valves described, such as a device comprising a plate 132 having a plurality of recesses 134 in which rotating components engage.

It would be possible for the cassette 16 and/or the incubator to be equipped with automatic means for checking the correct positioning of the cassette 16 in its first and second positions respectively. To this end, a magnet can be placed within the housing 90 inside the lower half-shell 96, the magnet appropriately placed on the tray for the first and second positions to be detected. It works with two Hall effect sensors.

FIG. 20 shows a cell culture system 180 comprising multiple incubators 12 and centrifuges 20 described above. Incubator 12 and centrifuge 20 are controlled by a computer system.

FIG. 21 is a flow chart showing the steps of a cell culture process according to the invention.

Prior to the process described below, cell culture cassette 16 is filled with cell culture medium using transverse tube 39 . After filling, this tube 39 is immediately closed, for example by welding. Cell culture cassette 16 is then frozen flat, ie, in a horizontal position for storage and subsequent use.

In the first step 182 of the process, a computer system is used to acquire and record culture parameters specific to the biological protocol. Inputs are made by the operator, and parameters to be entered are, for example, patient identification, cassette identification, and the like. To facilitate the entry of these parameters, the computer system can be equipped with a barcode reader and the cassettes can have barcodes that directly inform the computer system of the number and nature of the cassette. Cells to be cultured are then entered by barcode reading of personal identification data shown on the label of the container, such as a bag, or by manual entry via its touch screen.

In a second step 184, the computer system assigns a given incubator to the operator after checking incubator availability according to the cell culture schedule. An operator loads a cell culture cassette 16 containing cell culture medium into the designated incubator, preferably without access to the doors 22, 30 of other incubators 12, i.e. locked. .

In a third step 186, the process consists of thawing the culture medium contained in the flexible bag of the culture cassette as described above. To this end, the cell culture cassette 16, sealed within a sterile protective envelope, is placed in a second position on the support and agitation device 52 of the incubator designated by the computer system, namely the support structure 84 (FIGS. 3A and 3B). Attach to The operator closes both doors 22,30 and the computer system initiates the thawing procedure, automatically locking the doors 22,30. The support and agitation device is operated so that the cassette 16 is arranged horizontally.

In a fourth step 188, after thawing, the cell culture cassette 16 is removed from the incubator and the outer shell is removed with an appropriately controlled active area.

In a fifth step 190, cells of interest, excluding human embryonic stem cells, are injected into the cassette. To this end, manual valve 128 is placed in the open position. Recall that tubes 36, 38 are provided with non-return means to prevent the liquid in bag 100 from flowing out. Cells are injected into bag 100 via tube 38, then valve 128 is placed in the closed position and tube 38 is sealed with a tight cap.

In a sixth step 192, the cassette 16 filled with culture medium and cells of interest is loaded in its first position corresponding to the incubation position. A strip 34 is attached to the opening 32 of the door 22 and the end of the tube 36 opposite the bag 100 is passed through the opening 38 and connected to the fluid circuit of the system shown in FIG.

The process according to the invention then includes an incubation step 194 which can last several days, for example about 10 days. Periodically, the protocol parameters allow the contents of bag 100 to be homogenized by vibrating tray 54 about axis 63 as described above. This homogenization (duration, repetition frequency, amplitude) is determined by the protocol parameters chosen.

During the incubation step, the operator can remove one or more samples from bag 100 (196) (FIGS. 14 and 22). Some of these samplings can be offloaded to the computer system. Samples are removed using tubes 36a and 36b.

In removing the first sample, the computer system actuates movable member 136 associated with its corresponding valve 130a to allow fluid to pass through tube 36a. If sampling is performed by a computer system, a final filling operation of tube 36a is performed to ensure sterility and avoid any subsequent use of sampling tubes that have already been used. A second sample is taken in the same manner but with tube 36b.

After each sub-step of sampling 196 from the bag 100, the operator can perform an analysis of the sample and the results can be entered into a computer system and recorded by the operator or automatically. .

In the final step of process 198, the computer system retrieves or drains the contents of bag 100 of cassette 16. FIG.

Finally, the contents of the bag are subjected to purification steps and filling into sterile syringes for later use.

The present application also relates to cell culture processes, particularly mammalian cells, particularly human cells, with the exception of human embryonic stem cells.

These cells, in particular human cells other than human embryonic stem cells, may advantageously be or comprise (human) CD34+ cells, in particular (human) CD34+ hematopoietic cells or (human) CD34+ non-hematopoietic cells. It's okay. These (human) CD34+ cells can be, for example, cells from (human) peripheral blood, (human) umbilical cord, or human tissue, in particular (human) peripheral blood.

These cells, in particular human cells, with the exception of human embryonic stem cells, may advantageously be or comprise stem cells or progenitor cells, in particular multipotent or pluripotent cells.

These cells are not and do not contain human embryonic cells.

These cells, particularly human cells, with the exception of human embryonic stem cells, are advantageously peripheral blood human CD34+ (multipotent or pluripotent) mobilized by growth factors such as G-CSF (growth colony stimulating factor). They may be or include stem cells or progenitor cells.

These cells, particularly human cells other than human embryonic stem cells, may be or may be taken from human subjects or patients (neonatal, pediatric, adolescent, adult, or geriatric). The present invention is particularly adapted to human CD34+ (multipotent or pluripotent) cells that can be harvested from the peripheral blood of human subjects or patients. If the patient is undergoing chemotherapy, these cells may have been taken before or at the beginning of this chemotherapy.

A biological sample containing cells for culture (e.g. a sample of (human) blood, in particular (human) peripheral blood, (human) umbilical cord blood, or a sample of human tissue, in particular a sample of human peripheral blood) is a human subject. Or is or has been taken from a patient.

The means of the present application have the advantage that they do not require the use of leukapheresis: a simple blood sample, for example a sample of 200-250 mL blood, especially 220 mL of blood, can produce a significant number of cells, especially therapeutic and/or prophylactic treatment, in particular autologous or allogeneic cell therapy of a (human) subject or patient, in particular autologous cell therapy of a (human) subject or patient.

This applies in particular to human CD34+ cells for therapeutic and/or prophylactic treatment, in particular autologous or allogeneic cell therapy of a subject or patient, in particular autologous cell therapy of a subject or patient. Human CD34+ cells can be human non-embryonic CD34+ cells, excluding human embryonic stem cells. Human CD34+ cells are not and do not include embryonic CD34+ cells.

In the cell culture means of the present application, at least 10 ⁷ cells containing at least 70% human (non-embryonic) CD34+ cells or A population of at least 10 ⁸ cells can be obtained. In contrast, to obtain such amounts of human CD34+ cells directly (without cell culture) from the blood of a single subject or patient, an amount of 7-10 L of blood needs to be treated by leukapheresis. there will be

Cells for culture, particularly human (non-embryonic) CD34+ cells, may be obtained from biologically collected blood samples, particularly peripheral blood samples (e.g., 200-250 mL of peripheral blood, particularly 220 mL of peripheral blood). can be purified or isolated from a target sample.

Cells can be purified to achieve minimal levels of specific cell types. For example, the cells are purified to contain at least 70%, or at least 80%, or at least 85%, or at least 90% human (non-embryonic) CD34+ cells, particularly human CD34+ hematopoietic (non-embryonic) cells. can be done.

This isolation or purification can be done using any means known to any person skilled in the art.

For example, for human CD34+ cells, excluding human embryonic stem cells, this isolation or purification is performed using a human anti-CD34 monoclonal antibody, e.g. ) using a human mouse anti-CD34 monoclonal antibody sold under the name of (Human) CD34+ cell isolation or purification system, e.g. CD34 microbead kits are also commercially available.

Purified or isolated cells are placed on or in a culture medium whose components are compatible with the growth of these cells. Examples of such culture media, including media suitable for culturing human CD34+ cells, are described above. For example, such a culture medium can be a culture medium as described above, including medium containing human insulin, human transferrin, and human plasma or serum. This culture medium can be included in the bag of the cassette of the present application.

The cells are then incubated, for example, by placing the bag or cassette containing the bag in an incubator containing an incubator according to the present application. For example, the incubation time can be several days, especially 8-10 days, especially 9 days, especially when it concerns the culture of human CD34+ cells, excluding human embryonic stem cells.

The present application also relates to cells other than human embryonic stem cells, cell populations comprising such cells, and pharmaceutical compositions comprising such cells or cell populations.

These cells or cell populations can be obtained by culturing according to the present application, as described above.

In the present application, cells may in particular comprise (human) CD34+ cells, in particular (human) CD34+ hematopoietic cells or (human) CD34+ non-hematopoietic cells, excluding human embryonic stem cells.

These (human) CD34+ cells can be, for example, cells from (human) peripheral blood, (human) umbilical cord, or human tissue, in particular (human) peripheral blood.

These cells may advantageously be or comprise stem or progenitor cells, in particular multipotent or pluripotent cells, excluding human embryonic stem cells.

These cells are not and do not contain human embryonic cells.

These cells may in particular comprise human CD34+ (multipotent or pluripotent) stem or progenitor cells from peripheral blood that are mobilized by growth factors such as G-CSF (growth colony stimulating factor).

The cells of the application, including the (human) CD34+ cells of the application, excluding human embryonic stem cells, may be in pure or isolated form, or included in cell populations.

Accordingly, a cell population according to the application may be a population of human non-embryonic cells comprising human CD34+ cells according to the application.

In particular, cells according to the present application may comprise (a population of) human non-embryonic cells, said human non-embryonic cells having the following characteristics:
- said human non-embryonic cells are at least 10 ⁷ human cells, in particular at least 10 ⁸ or at least 1.5 x 10 ⁸ human cells, or between 10 ⁷ and 1.7 x 10 ⁸ human cells; The human non-embryonic cells are at least 70% human CD34+ (multipotent or pluripotent and non-embryonic) cells, in particular at least 80%, or at least 85%, or at least 90% human CD34+ (multipotent or pluripotent and non-embryonic) cells.

These human cells may also all have the same HLA types, in particular the same HLA-A, HLA-B, HLA-C and HLA-DR types.

For example, cells according to the present application may comprise (a population of) human non-embryonic hematopoietic cells, wherein said human non-embryonic hematopoietic cells are characterized by:
- said human non-embryonic hematopoietic cells are at least 10 ⁷ human non-embryonic hematopoietic cells, in particular at least 10 ⁸ , or at least 1.5×10 ⁸ , or 10 ⁷ to 1.7×10 ⁸ human non-embryonic hematopoietic cells are hematopoietic cells, and said human non-embryonic hematopoietic cells are at least 70% human CD34+ (multipotent or pluripotent and non-embryonic) cells, in particular at least 80%, or at least 85%, or including containing at least 90% human CD34+ (multipotent or pluripotent and non-embryonic) cells.

These human hematopoietic cells may also all have the same HLA type, in particular the same HLA-A, HLA-B, HLA-C and HLA-DR types.

Cells of the present application may have one or more characteristics that distinguish them from naive cells.

The term "naive" in this context means that these are cells obtained directly from a human subject or patient, and cells that may have been purified but have not been cultured.

Indeed, after culturing in accordance with the present application (e.g., on culture media and/or in bags or cassettes as described above), the resulting human cell population is initially cultured naive population is defined as: point:
a larger average diameter (diameter of 11.2±0.5 μm, especially 11.18±0.49 μm),
a high percentage of CD34-CD14+ cells (monocytes that may have a positive impact on the efficiency of cell transplantation);
A high percentage (but not 100%) of CD34+CD33- cells (CD33 is a marker of myeloid lineage);
• A small proportion of CD34+CD133+ cells (a marker of endothelial progenitor cells) (still above 10%) has been observed to differ by one or more.

In particular, the cells of the present application can be human cells other than human embryonic stem cells, said human cells having the following characteristics:
1. said human cells are at least 10 ⁷ human cells, in particular at least 10 ⁸ or at least 1.5×10 ⁸ or between 10 ⁷ and 1.7×10 ⁸ human cells,
2. said human cells all have the same HLA type, in particular the same HLA-A type, HLA-B type, HLA-C type and HLA-DR type;
3. said human cells are at least 70% human CD34+ (multipotent or pluripotent and non-embryonic) cells, in particular at least 80%, or at least 85%, or at least 90% human CD34+ (multipotent or multipotent) cells; (potential and non-embryonic) cells;
Four. said human cells have a cell diameter of 11.2±0.5 μm, in particular 11.18±0.49 μm,
Five. said human cells comprise (human) CD34-CD14+ cells in a proportion of 1-12%, in particular 2-10% or 3.8-10%;
6. said human cells represent more than 10% (human) CD34+CD33- cells, or more than 20%, or more than 25% (and advantageously less than 100%, especially less than 60%, or less than 50%, or 40%) less than or less than 35%);
7. said human cells comprise (human) CD34+CD133+ cells in a proportion of less than 60% or less than 50% (and advantageously more than 20%, especially more than 25% or more than 30%). have more than

According to one embodiment, the cells of the present application exhibit at least 2 of these 7 characteristics, in particular at least 3, or at least 4, or at least 5, or at least 6 of these 7 characteristics, or human cells, excluding human embryonic stem cells, that have all seven characteristics.

For example, the cells of the present application can be human hematopoietic cells, said human hematopoietic cells having the following characteristics:
1. said human hematopoietic cells are at least 10 ⁷ human hematopoietic cells, in particular at least 10 ⁸ or at least 1.5×10 ⁸ or 10 ⁷ to 1.7×10 ⁸ human hematopoietic cells,
2. said human hematopoietic cells all have the same HLA type, in particular the same HLA-A type, HLA-B type, HLA-C type and HLA-DR type;
3. said human hematopoietic cells are at least 70% human CD34+ (multipotent or pluripotent and non-embryonic) cells, in particular at least 80%, or at least 85%, or at least 90% human CD34+ (multipotent or pluripotent and non-embryonic) cells;
Four. said human hematopoietic cells have a cell diameter of 11.2±0.5 μm, especially 11.18±0.49 μm,
Five. said human hematopoietic cells comprise (human) CD34-CD14+ cells in a proportion of 1-12%, in particular 2-10% or 3.8-10%;
6. said human hematopoietic cells comprise (human) CD34+CD33- cells by more than 10%, or by more than 20%, or by more than 25% (and advantageously by less than 100%, in particular by less than 60%, or by less than 50%, or by 40%) % or less than 35%);
7. said human hematopoietic cells comprise (human) CD34+CD133+ cells in a proportion of less than 60% or less than 50% (and advantageously more than 20%, in particular more than 25% or more than 30%) have one or more

According to one embodiment, the cells of the present application exhibit at least 2 of these 7 characteristics, in particular at least 3, or at least 4, or at least 5, or at least 6 of these 7 characteristics, or human hematopoietic cells with all seven characteristics.

In particular, the cells of the present application can be human cells other than human embryonic stem cells, said human cells having the following characteristics:
1. said human cells are at least 10 ⁷ human cells, in particular at least 10 ⁸ or at least 1.5×10 ⁸ or between 10 ⁷ and 1.7×10 ⁸ human cells,
2. said human cells all have the same HLA type, in particular the same HLA-A type, HLA-B type, HLA-C type and HLA-DR type;
3. said human cells are at least 70% human CD34+ (multipotent or pluripotent and non-embryonic) cells, in particular at least 80%, or at least 85%, or at least 90% human CD34+ (multipotent or multipotent) cells; (potential and non-embryonic) cells;
Four. said human cells have a cell diameter of 11.2±0.5 μm, in particular 11.18±0.49 μm,
Five. said human cells comprise (human) CD34-CD14+ cells in a proportion of 1-12%, in particular 2-10% or 3.8-10%.

According to one embodiment, the cells of the present application are human cells that have at least 2 of these 5 characteristics, in particular at least 3, or at least 4, or all 5 of these 5 characteristics. Human cells, excluding embryonic stem cells.

For example, the cells of the present application can be human hematopoietic cells, said human hematopoietic cells having the following characteristics:
1. said human hematopoietic cells are at least 10 ⁷ human hematopoietic cells, in particular at least 10 ⁸ or at least 1.5×10 ⁸ or 10 ⁷ to 1.7×10 ⁸ human hematopoietic cells,
2. said human hematopoietic cells all have the same HLA type, in particular the same HLA-A type, HLA-B type, HLA-C type and HLA-DR type;
3. said human hematopoietic cells are at least 70% human CD34+ (multipotent or pluripotent and non-embryonic) cells, in particular at least 80%, or at least 85%, or at least 90% human CD34+ (multipotent or pluripotent and non-embryonic) cells;
Four. said human hematopoietic cells have a cell diameter of 11.2±0.5 μm, especially 11.18±0.49 μm,
Five. said human hematopoietic cells comprise (human) CD34-CD14+ cells in a proportion of 1-12%, in particular 2-10% or 3.8-10%.

According to one embodiment, the cells of the present application are human cells that have at least 2 of these 5 characteristics, in particular at least 3, or at least 4, or all 5 of these 5 characteristics. Hematopoietic cells.

In particular, the cells of the present application can be human cells other than human embryonic stem cells, said human cells having the following characteristics:
- said human cells are at least 10 ⁷ human cells, in particular at least 10 ⁸ or at least 1.5 x 10 ⁸ human cells, or between 10 ⁷ and 1.7 x 10 ⁸ human cells, and - said human cells are , at least 70% human CD34+ (multipotent or pluripotent and non-embryonic) cells, in particular at least 80%, or at least 85%, or at least 90% human CD34+ (multipotent or pluripotent and non-embryonic) cells, and further characterized by:
- said human cells have a cell diameter of 11.2±0.5 μm, in particular 11.18±0.49 μm,
- said human cells comprise (human) CD34-CD14+ cells in a proportion of 1-12%, in particular 2-10% or 3.8-10%.

For example, the cells of the present application can be human hematopoietic cells, said human hematopoietic cells having the following characteristics:
- said human hematopoietic cells are at least 10 ⁷ human hematopoietic cells, in particular at least 10 ⁸ or at least 1.5 x 10 ⁸ , or between 10 ⁷ and 1.7 x 10 ⁸ human hematopoietic cells, and human hematopoietic cells are at least 70% human CD34+ (multipotent or pluripotent and non-embryonic) cells, in particular at least 80%, or at least 85%, or at least 90% human CD34+ (multipotent or multipotent) cells; comprising viable and non-embryonic) cells;
and further features:
- said human hematopoietic cells have a cell diameter of 11.2±0.5 μm, in particular 11.18±0.49 μm,
- said human hematopoietic cells comprise (human) CD34-CD14+ cells in a proportion of 1-12%, in particular 2-10% or 3.8-10% (or both).

All cells in this population can have the same HLA-A, HLA-B, HLA-C, and HLA-DR types.

Cells in this population also:
>10%, or >20%, or >25% (and advantageously less than 100%, especially less than 60%, or less than 50%, or less than 40%, or less than 35%) (humans) CD34+CD33- cells,
• It may also contain a proportion of (human) CD34+CD133+ cells of less than 60% or less than 50% (and advantageously more than 20%, in particular more than 25% or more than 30% of CD34+CD133+ cells).

Alternatively, or in addition, the cells of the present application may have one or more characteristics identical to (or not significantly different from) those of naive cells, particularly naive human cells, particularly naive human hematopoietic cells.

Indeed, after culturing according to the present application (e.g., on culture media and/or in bags of cassettes as described above), the resulting human cell population (including the resulting human hematopoietic cell population) is initially It may share features with those of cultured naive (hematopoietic) cell populations.

In particular, the cells of the present application have the following characteristics compared to naive cells (including naive hematopoietic cells), especially compared to initially cultured (naive) cells:
1. the same (or not significantly different) number or proportion of CD34 epitopes,
2. identical (or not significantly different) ability to divide, in particular telomeres of identical (or not significantly different) relative lengths;
3. the same absence of chromosomal aberrations and polyploidy (or the same chromosomal aberrations or polyploidy in the same proportion),
Four. identical (or not significantly different) viability, in particular identical (or not significantly different) CD34+ cell viability, such as at least 90% or at least 95% CD34+ cell viability;
Five. the same (or not significantly different) proportion of CD34-CD2+/CD3+ cells,
6. an identical (or not significantly different) proportion of CD34-CD19+/CD20+ cells,
7. the same (or not significantly different) proportion of CD34-CD56+ cells,
8. The cells (including hematopoietic cells) may have one or more of the same (or not significantly different) proportions of CD34-CD15+ cells.

According to a further embodiment, the cells of the present application exhibit at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7 of these eight characteristics, or have all eight characteristics.

In particular, the cells of the present application have the following characteristics compared to naive (hematopoietic) cells, especially compared to initially cultured (naive) cells:
1. the same (or not significantly different) number or proportion of CD34 epitopes,
2. identical (or not significantly different) ability to divide, in particular telomeres of identical (or not significantly different) relative lengths;
3. A cell (including a hematopoietic cell) having one or more of chromosomal aberrations and the same absence of polyploidy (or the same proportion of chromosomal aberrations or polyploidy).

According to a further embodiment, the cells of the present application have at least two or all three of these three characteristics.

This application:
- Characteristics of the cells of the present application that differ from naive cells,
• Explicitly includes any combination of cellular features among those described herein, including combinations of features that are identical to features of naive cells.

The cell culture means of the present application essentially correspond to ex vivo cell expansion means. This cell culture means produces a significant number (especially a number sufficient for treatment) of autologous or allogeneic cells, particularly autologous cells (especially autologous or allogeneic CD34+ cells, In particular, it has the advantage of providing autologous CD34+ cells) without detrimentally altering these cells.

Table 1 below shows cultures in culture bags according to the present application (IMDM medium (supplemented with glutamine, fibroblast of healthy human volunteers without cells, containing 0.01 mg/mL insulin, 330 pg/ml transferrin, and 5% (VN) human serum) for 9 days. An illustration of the population characteristics of cells obtained from peripheral blood of a cohort is shown:

Once the culture is complete, the obtained cells, in particular the obtained (human) CD34+ cells, can be harvested.

Harvested cells can be purified or isolated, in particular purified. In particular, CD34+ cells can be purified by using purification means available to those skilled in the art, eg, as described above.

The harvested and optionally purified cells can be included in compositions, particularly pharmaceutical compositions, particularly liquid pharmaceutical compositions, such as non-toxic pharmaceutical isotonic pharmaceutical solutions for humans. The resulting composition or solution is the subject of this application.

The composition may contain a vehicle that is physiologically acceptable for human use.

The composition can be formulated to be administrable, particularly injectable, to humans or into the human body. Thus, the composition may be liquid and contains a compound that is non-toxic to humans, preferably at isotonic concentrations for humans.

Thus, in addition to the cells, the composition may contain, for example, a non-toxic buffer for humans, such as phosphate-buffered saline (phosphate-buffered saline or PBS), or gluconate and/or acetate buffers. may contain liquid.

The composition may also contain at least one additive that is non-toxic to humans, such as proteins from human serum or human plasma, including human serum albumin (HSA).

Compositions or solutions of the present application can be stored, for example, by refrigeration (freezing) until use.

The means of the present application can be used in (therapeutic and/or prophylactic) cell therapy, in particular autologous or allogeneic cell therapy, advantageously autologous cell therapy.

This cell therapy:
- the cells of the present application, in particular human cells, including CD34+ cells excluding human embryonic stem cells as described above, or - pharmaceutical compositions or solutions comprising these cells, to the patient or subject.

This patient or subject may be the same patient or subject from whom the cells were originally harvested for cell culture (autologous cell therapy or therapy). Alternatively, the patient or subject may be different from the patient or subject from whom the cells were originally obtained for cell culture (autologous cell therapy or therapy).

Cell therapy, particularly autologous or allogeneic (especially autologous) cell therapy, may include treatment or prevention of heart failure or non-cardiac disease.

Heart failure is especially:
heart failure, including ventricular failure, especially left ventricular failure;
Heart failure, especially with systolic dysfunction,
especially heart failure with alterations in the contractile function of the ventricles, especially the left ventricle;
- More specifically, it may be heart failure with increased end-systolic volume associated with decreased left ventricular ejection fraction (LVEF).

This includes the New York Heart Association (NYHA) Classification [The Criteria Committee of the New York Heart Association]. Criteria for Diagnosis of Diseases of the Heart and Great Vessels. (9th ed.)”. Boston: Little, Brown & Co., pages 253-256]. Level II or higher (according to the NYHA classification) heart failure may be included.

For heart failure:
・High blood pressure,
・Valve disorder,
・Congenital diseases,
- Treatment with refractory angina (e.g., Enhanced Extracorporeal CounterPulse (EECCP) or nerve stimulation therapy (especially transcutaneous nerve stimulation (TNS) or spinal cord stimulation (SMS)), or chelation therapy (especially EDTA) angina pectoris that is not effective with administration therapy)),
especially cardiomyopathy,
Ischemic cardiomyopathy, especially myocardial infarction, especially acute myocardial infarction,
・dilated cardiomyopathy,
・Hypertrophic cardiomyopathy,
・Restrictive cardiomyopathy,
• Heart failure caused by arrhythmogenic right ventricular cardiomyopathy (ARVC) may be included.
Heart failure includes cardiomyopathy, especially ischemic cardiomyopathy, especially myocardial infarction, especially acute myocardial infarction,
・dilated cardiomyopathy,
・Hypertrophic cardiomyopathy,
・Restrictive cardiomyopathy,
• Heart failure caused by arrhythmogenic right ventricular cardiomyopathy (ARVC) may be included.

Heart failure can include heart failure caused by cardiomyopathy, especially ischemic cardiomyopathy, especially myocardial infarction, especially acute myocardial infarction.

Heart failure can be treated or prevented, inter alia, by alleviating or curing heart failure and/or by limiting the onset of heart failure.

Heart failure caused by ischemic cardiomyopathy, especially myocardial infarction, especially acute myocardial infarction, can be treated or prevented by alleviating or curing the heart failure and/or by limiting the onset of the heart failure. .

Indeed, the cells (especially CD34+ cells) obtained by culturing and optionally purification according to the present application, or pharmaceutical compositions or solutions comprising these cells, are intended for the following purposes:
- reduce or reverse post-ischemic myocardial damage, including post-infarction necrosis and/or post-infarction scar size;
- partially or completely regenerate the structure and/or regenerate the blood vessels of the myocardium or the infarcted area;
- improving or restoring ventricular function, in particular left ventricular function, in particular ventricular contractility, in particular left ventricular contractility, e.g. increasing left ventricular ejection fraction (LVEF) (especially achieving LVEF greater than 45%) reducing end-systolic volume in combination with

More specifically, it is intended for the treatment or prevention of heart failure caused by (acute) myocardial infarction, in particular by (acute) myocardial infarction due to coronary heart disease or heart attack. This use is specifically for:
- Patients with impaired left ventricular function or contractility, and - First event or cardiac syndrome (this first event or cardiac syndrome includes stent placement(s) and percutaneous transluminal coronary angioplasty (PTCA) and/or have a new-onset acute myocardial infarction following a coronary artery bypass graft (CABG)), and at least 10 days after this first cardiac event (e.g., at least Patients with a left ventricular ejection fraction (LVEF) of 45% or less after 3 months or at least 6 months.

Administration of the cells of the application, the cells of the pharmaceutical composition or solution of the application, or the pharmaceutical composition or solution of the application can occur, for example, within a few weeks, particularly within 3-6 weeks, of the ischemic stroke.

Treatment or prevention of heart failure may include cellular cardiomyoplasty (by transplanting administered cells into an infarcted heart area).

Such treatment or prevention includes cell populations according to the present application (including populations of human cells comprising CD34+ cells excluding human embryonic stem cells), cells according to the present application (including human CD34+ cells excluding human embryonic stem cells), or administration of compositions or solutions according to the present application by surgery or infusion thereof through a catheter.

Surgical injection can be direct injection into the infarct area, for example during coronary artery bypass grafting (CABG).

Catheter injection may include percutaneous femoral injection.

Catheters may include intracoronary, epicardial, or transendocardial injection or infusion catheters.

For example, for injection or infusion catheters, BIOCARDIA® (125 Shoreway Road, Suite B, San Carlos, CA 94070, U.S.A.) can be coupled to a two-dimensional (2D) guidance system, such as a 2D fluoroscopic guidance system. can be equipped with a helical needle, such as the HELIX® catheter marketed by Co., Ltd.).

The injection or infusion catheter can be, for example, the MYOSTAR® catheter sold by BIOSENSE WEBSTER, INC. (15715 Arrow Highway; Irwindale; CA 91706; U.S.A.).

The injection or infusion catheter can be attached to a three-dimensional (3D) guidance system such as, for example, the NOGA® XP Electromagnetic 3D Cardiac Mapping System sold by BIOLOGICS DELIVERY SYSTEMS GROUP (CORDIS Corp., Diamond Bar, Calif., U.S.A.). can be combined.

One or more chemokines, particularly one or more CXC family chemokines, particularly chemokine CXCL12 (stromal cell-derived factor 1 or SDF-1), may be administered, particularly injected, to a patient or subject. Such administration or injection includes cell populations according to the application (including populations of human cells including CD34+ cells excluding human embryonic stem cells), cells of the application (including human CD34+ cells excluding human embryonic stem cells), or Administration, particularly injection, of the compositions or solutions of the present application may be simultaneous with, subsequent to, or delayed in time.

The use of the means of the present application is not limited to the treatment or prevention of heart failure caused by ischemic cardiomyopathy or heart failure caused by chemotherapy. The means of the present application (especially the CD34+ means of the present application) can alternatively be used to treat or prevent non-cardiac disease.

Non-cardiac disease can be a non-cardiac condition not induced by a cardiac procedure.

The means of the present application can be used to treat myelosuppression, including myelosuppression induced by the treatment of lymphoma, particularly non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia. Then, using the means of the present application, hematopoietic cells (either autologous or derived from another subject), including populations of hematopoietic cells (autologous or allogeneic) comprising CD34+ cells, are intravenously transplanted as described above. It can be transplanted into the patient's bone marrow. The transplanted cells are then used to regenerate the patient's bone marrow.

Alternatively, the means of the application can be used to treat or prevent conditions that induce cartilage degeneration such as osteoarthritis. The means of the application can then be used to transplant cells according to the application (either autologous or derived from another subject), including a cell population comprising CD34+ cells, as described above, into a patient, particularly an area of arthritis. can. The implanted cells are then used to regenerate the patient's cartilage.

Alternatively, the means of the present application can be used to treat or prevent liver failure, especially chronic liver failure, especially chronic non-alcoholic liver failure (eg, to prevent the development of hepatocellular carcinoma). Then, using the means of the present application, cells according to the present application (autologous or derived from another subject), comprising a population of cells (autologous) comprising CD34+ cells, as described above, are introduced into the patient, particularly in the region of the liver. can be transplanted. The transplanted cells are then used to regenerate the patient's liver.

The term "including", synonymous with "including" or "containing," is an open-ended term and includes one or more additional While not excluding the presence of element(s), component(s), or process step(s), the terms "consisting of" or "consisting of" are terms of limitation and express excludes the presence of any other additional element, step, or ingredient not specifically mentioned. The terms "consisting essentially of" or "consisting essentially of" are partial open-ended terms that include one or more additional element(s), component(s), or The presence of such additional element(s), ingredient(s), or step(s) is disregarded if the step(s) do not materially affect the essential nature of the invention. not excluded.

Thus, the term "including" (or "includes") is used to refer to the terms "consisting of", "consisting of", as well as "consisting essentially of" and "consisting essentially of". including the term "target".
The present application provides inventions of the following aspects.
(Aspect 1)
- a housing (90) that is at least partially rigid and defines an interior space therein in which a cell culture bag (100) defining an interior volume is positioned and secured;
- conduits (36, 38) each connected at one end to the interior volume of the bag (100) and each having a second end located outside the housing (90);
• A cell culture cassette (16) comprising valves (128, 130) for passing/blocking fluid flow through said conduits (36, 38) attached to said housing (90).
(Aspect 2)
The conduits (36, 38) are formed by flexible tubes, and at least some of the valves (128, 130) route the tubes to a structural member (132) of the housing (90). The cassette of aspect 1, comprising movable members (136, 138) for tightening.
(Aspect 3)
Each moveable member (136, 138) has an axis of rotation (136c, 138c), and the radial circumference of each moveable member (136, 138) rotates along the tube (136, 138) as the moveable member (136, 138) rotates. at least one helical surface (160) about said axis of rotation (136c, 138c) forming a clamping surface of said tube (36, 38) to vary the fluid passage cross-section of said tube (36, 38); A cassette according to embodiment 2.
(Aspect 4)
The movable members (136, 138) are disposed within the housing (90) and mounted in recesses (134) of a plate (132) incorporated in the housing (90), each recess (134) being movable. 4. The cassette of embodiment 3, which receives a member.
(Aspect 5)
Each movable member (136, 138) comprises an end (136a, 138b) accessible from outside said housing (90), said end (136a, 138b) connecting to the second rotary coupling means of the actuator. A cassette according to any one of aspects 2-4, comprising a first rotational coupling means (154) cooperating with.
(Aspect 6)
Aspect 4 or aspect 4 and characterized in that said movable part is centrally guided by rotation so that one end thereof is in the opening of the housing and the opposite end is in the opening of the plate. 5 described cassette.
(Aspect 7)
The bag (100) comprises a first flexible wall (122) and a second flexible wall that are substantially parallel to each other in both an empty state and a liquid filled state. A cassette according to any one of aspects 2-6, having a rectangular shape.
(Aspect 8)
The ratio of the sum of the surface of said first wall (122) and said second wall to the total internal volume of said bag (100) is 500-690 cm 2 /L, preferably about 580 cm ² / L ^. A cassette according to aspect 7.
(Aspect 9)
A cassette according to aspect 7 or 8, wherein the distance between said first wall (122) and said second wall is less than 20 mm, preferably between 10 and 20 mm, more preferably about 14 mm.
(Mode 10)
10. Any of the embodiments 1 to 9 , wherein the walls of said bag (100) are permeable to gases, in particular CO2, preferably with properties that limit as much as possible the adhesion of cells to the walls of said bag (100) _. Or the cassette described in one item.
(Aspect 11)
The housing (90) comprises a lower half-shell (96) and an upper half-shell (98) fixed to each other and having a shape substantially corresponding to the shape of a rectangular parallelepiped, the lower half-shell (96) ) is rigid and said upper half-shell (98) is rigid or flexible.
(Aspect 12)
The upper half-shell (98) allows propagation of a wave of liquid to the opening (108) of the pocket (100) when the cassette (16) containing liquid is vibrated about a horizontal axis. 12. The cassette of aspect 11, comprising the opening (108) or central recess shaped and sized as such, the opening being positioned upward.
(Aspect 13)
Cassette according to aspect 11 or 12, wherein said lower half-shell (96) preferably comprises a plurality of holes (104), for example with a diameter of less than 20 mm.
(Aspect 14)
Aspect 11, wherein said conduits (36, 38) extend through one of the outer perimeter edges (102a, 102b) of one of said lower half-shell (96) or said upper half-shell (98). 14. The cassette according to any one of -13.
(Aspect 15)
15. Any one of aspects 1-14, comprising a conduit (36, 38) support strip (34) located external to said housing (90) and traversed by at least some of said conduits (36, 38) The cassette described in the paragraph.
(Aspect 16)
A cassette according to any one of aspects 1 to 15, characterized in that said bag contains cell culture medium.
(Aspect 17)
17. A cassette according to embodiment 16, characterized in that said cell culture medium comprises human insulin, human transferrin and human plasma or serum.
(Aspect 18)
A cassette according to aspect 16 or 17, characterized in that the cell culture medium is frozen.
(Aspect 19)
A cassette according to any one of aspects 1 to 17, characterized in that said bag contains CD34+ cells.
(Aspect 20)
A thermostatic enclosure (28) and a device (14) for supporting and agitating the cell culture cassette of any one of aspects 1-19, and placing said cell culture cassette (16) in said support device. A cell culture incubator comprising means for placing and locking in position.
(Aspect 21)
Said support and agitation device (14) comprises a tray (54) for supporting a cell culture cassette (16) pivotally mounted about a horizontal axis (63), said tray (54) 21. An incubator according to embodiment 20, adapted to vibrate about said horizontal axis (63) to agitate and homogenize the contents of said bag (100).
(Aspect 22)
Said supporting and stirring device (14) comprises a connecting rod (64), one upper end of which is connected to said plate (54) via an oscillator, and a lower end of said connecting rod is connected to its axis. 22. An incubator according to aspect 21, wherein (70) is connected to a crank (68) rotationally driven by a substantially horizontal motor shaft.
(Aspect 23)
The tray (54) holds an electromechanical actuator (82) for opening and closing the valves (128, 130), one output of the electromechanical actuator activating the cassette (16). 23. An incubator according to aspect 21 or 22, holding second coupling means cooperating with first coupling means (154) of said valves (128, 130) of said cassette when in a given position.
(Aspect 24)
a door (22) for sealing an opening (26) of said enclosure (28), said opening (26) having a peripheral edge (24) with a recess (32) formed therein, said recess The shape of (32) is adapted to receive said support strip (34) of a conduit of a cassette according to aspect 15, said strip (34) being engaged when said door is in the closed position of said enclosure (28). 24. An incubator according to any one of aspects 20 to 23, wherein a sealing member is formed between said door (22) and said peripheral edge.
(Aspect 25)
25. An incubator according to any one of aspects 20-24, comprising means for controlling the position of said cell culture cassette (16) in said support means.
(Aspect 26)
At least one incubator (12) according to any one of aspects 20 to 25 and for regulating culture conditions in an enclosure of said at least one incubator and controlling valves of said cassette housed in said enclosure and a computer control system including data acquisition and recording means for cell culture automation.
(Aspect 27)
(a) placing a cell culture cassette (16) according to any one of embodiments 1-19 on said support and agitation device (14) in a thawed position within said incubator enclosure (12);
(b) supplying cells to be cultured to said bag (100);
(c) placing the cassette (16) on the support and agitation device (14) in a cell culture position;
(d) agitating said cassette (16) for a period of time to homogenize its contents;
(e) maintaining said cassette (16) under incubation conditions for several days; and
27. A method for culturing cells with an automated device according to embodiment 26, characterized by the step of: (f) recovering the contents of said cassette (16) by one of the tubes of said cassette (16).
(Aspect 28)
During step (d), including one or more sampling steps (196) for sampling the contents of said bag, wherein prior to each of said one or more steps said support tray (54) is placed horizontally. 28. A process according to embodiment 27, characterized in that from the incubation position the step of tilting the bag (100) to a tilted position where the sampling area of the bag (100) is the lowest point of the bag (100) is performed.
(Aspect 29)
29. Process according to embodiment 27 or 28, characterized in that said cells to be cultured are CD34+ cells.
(Aspect 30)
A pharmaceutical composition for use in the treatment of heart failure caused by myocardial infarction, comprising a cassette according to any one of aspects 1-19, or an incubator according to any one of aspects 20-25, or aspect 26. comprising cells that can be produced by culturing in an automated device as described or by the cell culturing process of any one of aspects 27-29, wherein said cells:
- are human cells;
- at least 10 ⁷ cells,
- Contains at least 70% human CD34+ cells,
・Contains 1-12% of human CD34-CD14+ cells, and
• Said pharmaceutical composition, characterized in that it has a cell diameter of 11.2±0.5 μm.

Claims (29)

- a housing (90) that is at least partially rigid and defines an interior space therein in which a cell culture bag (100) defining an interior volume is positioned and secured;
- conduits (36, 38), each connected at one end to the interior volume of the bag (100) and each having a second end located outside the housing (90);
- valves (128, 130) for passing/blocking fluid flow through said conduits (36, 38) attached to said housing (90);
a cell culture cassette (16). The conduits (36, 38) are formed by flexible tubes, and at least some of the valves (128, 130) route the tubes to a structural member (132) of the housing (90). The cassette of claim 1, comprising movable members (136, 138) for tightening. Each moveable member (136, 138) has an axis of rotation (136c, 138c), and the radial circumference of each moveable member (136, 138) rotates along the tube (136, 138) as the moveable member (136, 138) rotates. at least one helical surface (160) about said axis of rotation (136c, 138c) forming a clamping surface of said tube (36, 38) to vary the fluid passage cross-section of said tube (36, 38); 3. The cassette of claim 2. The movable members (136, 138) are disposed within the housing (90) and mounted in recesses (134) of a plate (132) incorporated in the housing (90), each recess (134) being movable. 4. The cassette of claim 3, which receives the member. Each movable member (136, 138) comprises an end (136a, 138b) accessible from outside said housing (90), said end (136a, 138b) connecting to the second rotary coupling means of the actuator. Cassette according to any one of claims 2 to 4, comprising a first rotary coupling means (154) cooperating with. 4. The movable member is guided centrally by rotation so that one end of the movable member is in the opening of the housing and the opposite end is in the opening of the plate (132) . The cassette according to 4 or 5 . The bag (100) comprises a first flexible wall (122) and a second flexible wall that are substantially parallel to each other in both an empty state and a liquid filled state. Cassette according to any one of claims 2 to 6, having a rectangular shape. The ratio of the sum of the surfaces of said first flexible wall (122) and said second flexible wall to the total internal volume of said bag (100) is 500-690 cm ² /L, preferably 580 cm 8. The cassette of claim 7, which is on the order of ² /L. Claim 7, wherein the distance between said first flexible wall (122) and said second flexible wall is less than 20 mm, preferably between 10 and 20 mm, more preferably about 14 mm. Or the cassette according to 8. 10. The method of claims 1 to 9, wherein the walls of the bag (100) are permeable to gases, in particular _CO2 , and preferably have properties that limit the adhesion of cells to the walls of the bag (100) as much as possible. A cassette according to any one of the preceding claims. The housing (90) comprises a lower half-shell (96) and an upper half-shell (98) fixed to each other and having a shape substantially corresponding to the shape of a rectangular parallelepiped, the lower half-shell (96) ) is rigid and the upper half-shell (98) is rigid or flexible. The upper half-shell (98) allows propagation of waves of liquid from the bag (100) to the opening (108) when the cassette (16) containing liquid is vibrated about a horizontal axis. 12. A cassette according to claim 11, comprising the opening (108) or central recess shaped and sized to allow the opening to face upwardly. Cassette according to claim 11 or 12, wherein the lower half-shell (96) comprises a plurality of holes (104), preferably with a diameter of less than 20 mm, for example. The conduit (36, 38) extends through one of the perimeter edges (102a, 102b) of one of the lower half-shell (96) or the upper half-shell (98). A cassette according to any one of 11-13. 15. Any of claims 1 to 14, comprising a conduit (36, 38) support strip (34) located outside said housing (90) and traversed by at least some of said conduits (36, 38). A cassette according to paragraph 1. Cassette according to any one of claims 1 to 15, characterized in that the bag contains cell culture medium. 17. Cassette according to claim 16, characterized in that said cell culture medium comprises human insulin, human transferrin and human plasma or serum. 18. Cassette according to claim 16 or 17, characterized in that the cell culture medium is frozen. Cassette according to any one of claims 1 to 17, characterized in that said bag contains CD34+ cells. A thermostatic enclosure (28) and a device (14) for supporting and agitating a cell culture cassette according to any one of claims 1 to 19, and said cell culture cassette (16) in said supporting and agitating device. A cell culture incubator comprising means for positioning and locking in a given position corresponding to a cell culture position within. Said support and agitation device (14) comprises a tray (54) for supporting a cell culture cassette (16) pivotally mounted about a horizontal axis (63), said tray (54) 21. An incubator according to claim 20, adapted to oscillate about said horizontal axis (63) to agitate and homogenize the contents of said bag (100). Said supporting and stirring device (14) comprises a connecting rod (64), one upper end of which is connected to said tray (54) via an oscillator, and a lower end of said connecting rod is connected to its axis. 22. The incubator of claim 21, wherein (70) is connected to a crank (68) rotationally driven by a substantially horizontal motor shaft. The tray (54) holds an electromechanical actuator (82) for opening and closing the valves (128, 130), one output of the electromechanical actuator activating the cassette (16). 23. An incubator according to claim 21 or 22, holding second coupling means cooperating with the first coupling means (154) of the valves (128, 130) of the cassette when in a given position. a door (22) for sealing an opening (26) of said enclosure (28), said opening (26) having a peripheral edge (24) with a recess (32) formed therein, said recess The shape of (32) is adapted to receive said support strip (34) of the conduit of the cassette according to claim 15, said strip (34) being in the closed position of said door in said enclosure (28). An incubator according to any one of claims 20 to 23, sometimes forming a sealing member between said door (22) and said peripheral edge. An incubator according to any one of claims 20 to 24, comprising means for controlling the position of said cell culture cassette (16) in said support and agitation device . At least one incubator (12) according to any one of claims 20 to 25 and for regulating culture conditions in an enclosure of said at least one incubator and controlling valves of said cassette housed in said enclosure. and a computer control system including data acquisition and recording means for performing. (a) placing a cell culture cassette (16) according to any one of claims 1 to 19 on said support and agitation device (14) in a horizontal thawed position within said incubator enclosure (12); the step of
(b) supplying cells to be cultured to said bag (100);
(c) placing said cassette (16) on said support and agitation device (14) in a cell culture position;
(d) agitating said cassette (16) for a period of time to homogenize its contents;
(e) maintaining the cassette (16) under incubation conditions for several days; and (f) collecting the contents of the cassette (16) by one of the tubes of the cassette (16). 27. A cell culture method using the automatic device according to claim 26, characterized in that: During step (d), including one or more sampling steps (196) for sampling the contents of said bag, prior to each of said one or more steps, removing said tray (54). 28. Method according to claim 27, characterized in that the step of tilting the bag (100) from a horizontal incubation position to a tilted position where the sampling area of the bag (100) is the lowest point of the bag (100) is carried out. 29. Method according to claim 27 or 28, characterized in that the cells to be cultured are CD34+ cells.

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